The interplay of insulin, cortisol and prolactin induces the differentiation of the mouse mammary gland in culture. Addition of progesterone (Prog) above 30 nM caused progressive inhibition of milk-protein synthesis and spermidine accumulation. The maximal degree of inhibition, which was attained at 300 nM, was about 40% for casein synthesis, 90% for alpha-lactalbumin synthesis and 70% for spermidine accumulation. The increase in the activity of S-adenosylmethionine decarboxylase and spermidine synthase, two key enzymes in polyamine biosynthesis, was inhibited by 55% and 65%, respectively, by Prog. The extent of Prog inhibition of milk-protein synthesis was greatest in cultured tissue from virgin mice, which contained a low level of spermidine, whereas mammary explants from lactating mice, which contained a high level of spermidine, was insensitive to Prog. The inhibitory effect of Prog can be partially antagonized by cortisol which causes an enhancement of spermidine accumulation that, in turn, mediates some action of cortisol on milk-protein synthesis. These results suggest that the synthesis and accumulation of spermidine serve as a target site for the inhibitory action of Prog on lactogenesis.